Project Summary: In this proposal, we will develop a synthetic nucleosome-based drug-screening platform to identify novel therapeutics to treat neuroblastoma. Histone acetylation is associated with gene activation and is catalyzed by histone acetyltransferase enzymes (HATs). Histone hyperacetylation is a key driver of neuroblastoma. Several recent studies demonstrate that inhibition of HAT activity dramatically slows cancer progression in vivo. However, there are no FDA approved HAT inhibitors for human use. New HAT inhibitors therefore, are greatly needed to develop therapies for hyperacetylation-based diseases such as neuroblastoma. Nucleosomes, composed of the core histone proteins and DNA, are the fundamental repeating units of chromatin. Chromatin structure and function are altered upon the addition or removal of histone post- translational modifications (PTMs), such as methylation, acetylation, and phosphorylation by histone modifying enzymes. The ?histone code hypothesis? stipulates that nucleosomal PTMs function in interdependent networks to regulate downstream gene expression. Current histone modifying assays typically use modified histone proteins/fragments as substrates, which poorly mimic native chromatin architecture. By contrast, synthetic nucleosomes carrying specific PTMs (termed ?designer nucleosomes? or ?dNucs?) provide a superior substrate for the study of histone modifying enzymes by better replicating chromatin structure. EpiCypher is a world leader in recombinant nucleosome synthesis and is pioneering the development of dNuc-based technologies for drug discovery applications. Phosphorylation of histone H3 at serine 10 (H3S10ph) is strongly associated with gene activation and acts as an epigenetic signaling hub that significantly enhances the activity of multiple HATs. In this Phase I proposal, we will leverage this unique feature of the histone code to develop an innovative screening platform to identify HAT inhibitors. We hypothesize that screening HAT enzymes in the presence of H3S10ph will recapitulate in vivo activity and reveal context-dependent inhibitors, providing a robust and powerful assay platform for drug discovery. We will develop for the first time methods to synthesize high quality H3S10ph- modified nucleosomes at commercial-grade and -scale. We will then use these dNucs as biochemical substrates to establish HAT activity assays using H3S10ph-dependent enzymes. Finally, we will demonstrate feasibility that this assay platform can be used for drug discovery, by examining phosphorylation context- dependent HAT activity following treatment of H3S10ph-modified nucleosomes with tool HAT inhibitors. In Phase II, we will further optimize commercialization of H3S10ph-modified nucleosomes to support high throughput assay development. We will also develop additional H3S10ph-dependent HAT activity/inhibitor assays, which we will market as stand-alone inhibitor kits to both industrial and academic research customers. The innovative drug discovery platform described herein will accelerate the identification HAT inhibitors to treat devastating human diseases such as neuroblastoma.